Method for sterilizing products

ABSTRACT

A method for sterilizing products to inactivate biological contaminants such as viruses, bacteria, yeasts, molds, mycoplasmas and parasites is disclosed. The method involves irradiating the product at a low dose rate from about 0.1 kGy/hr. to about 3.0 kGy/hr. for a period of time sufficient to sterilize the product. The method does not destroy sensitive materials such as blood and blood components. Further, the method does not require pre-treatment of the product such as freezing, filtration or the addition of chemical sensitizers.

[0001] This application is a continuation in part of PCT/CA94/00401filed Jul. 22, 1994 which is a continuation-in-part of U.S. Ser. No.08/095,698 filed Jul. 22, 1993, now U.S. Pat. No. 5,362,442.

FIELD OF THE INVENTION

[0002] The present invention relates to a method for sterilizingproducts to inactivate biological contaminants such as viruses,bacteria, yeasts, molds, mycoplasmas and parasites.

BACKGROUND OF THE INVENTION

[0003] Several products that are prepared for human, veterinary orexperimental use may contain unwanted and potentially dangerouscontaminants such as viruses, bacteria, yeasts, molds, mycoplasmas andparasites. Consequently, it is of utmost importance that anybiologically active contaminant in the product be inactivated before theproduct is used. This is especially critical when the product is to beadministered directly to a patient, for example in blood transfusions,organ transplants and other forms of human therapies. This is alsocritical for various biotechnology products which are grown in mediawhich contain various types of plasma and which may be subject tomycoplasma or other viral contaminants.

[0004] Previously, most procedures have involved methods that screen ortest products for a particular contaminant rather than removal orinactivation of the contaminant from the product. Products that testpositive for a contaminant are merely not used. Examples of screeningprocedures include the testing for a particular virus in human bloodfrom blood donors. However, such procedures are not always reliable andare not able to detect the presence of viruses in very low numbers. Thisreduces the value or certainty of the test in view of the consequencesassociated with a false negative result. False negative results can belife threatening in certain cases, for example in the case of AcquiredImmune Deficiency Syndrome (AIDS). Furthermore, in some instances it cantake weeks, if not months, to determine whether or not the product iscontaminated.

[0005] More recent efforts have focused on methods to remove orinactivate contaminants in the products. Such methods include heattreating, filtration and the addition of chemical inactivants orsensitizers to the product. Heat treatment requires that the product beheated to approximately 60° C. for about 70 hours which can be damagingto sensitive products. Heat inactivation can destroy up to 50% of thebiological activity of the product. Filtration involves filtering theproduct in order to physically remove contaminants. Unfortunately thismethod may also remove products that have a high molecular weight.Further, in certain cases small viruses may not be removed by the filterbecause of the larger molecular structure of the product. The procedureof chemical sensitization involves the addition of noxious agents whichbind to the DNA/RNA of the virus and which are activated either by UV orionizing radiation to produce free radicals which break the chemicalbonds in the backbone of the DNA/RNA of the virus or complex it in sucha way that the virus can no longer replicate. This procedure requiresthat unbound sensitizer is washed from cellular products since thesensitizers are toxic, if not mutagenic or carcinogenic, and can not beadministered to a patient.

[0006] Irradiating a product with gamma irradiation is another method ofsterilizing a product. Gamma irradiation is effective in destroyingviruses and bacteria when given in high total doses. (Keathly, J. D. Etal.; Is There Life after Irradiation? Part 2; BioPharm July-August,1993, and Leitman, Susan F.; Use of Blood Cell Irradiation in thePrevention of Post Transfusion Graft-vs-Host Disease; TransfusionScience 10:219-239, 1989) However, the published literature in this areateaches that gamma irradiation can be damaging to radiation sensitiveproducts such as blood. In particular, it has been shown that highradiation doses are injurious to red cells, platelets and granulocytes(Leitman, ibid). Van Duzer, in U.S. Pat. No. 4,620,908 discloses thatthe product must be frozen prior to irradiation in order to maintain theviability of a protein product. Van Duzer concludes that:

[0007] “If the gamma irradiation were applied while the protein materialwas at, for example, ambient temperature, the material would be alsocompletely destroyed, that is the activity of the material would berendered so low as to be virtually ineffective.”

[0008] Unfortunately, many sensitive biologicals, such as blood, wouldlose viability and activity if subjected to freezing for irradiationpurposes and then thawing prior to administration to a patient.

SUMMARY OF THE INVENTION

[0009] In view of the above, there is a need to provide a method ofsterilizing products that is effective in removing biologicalcontaminants while at the same time having no adverse effect on theproduct. The present invention has shown that if the irradiation isdelivered at a low dose rate, then sterilization can be achieved withoutharming the product. No prior references have taught or suggested thatapplying gamma irradiation at a low dose rate can overcome the problemsadmitted in the prior references.

[0010] Accordingly, the present invention provides a method forsterilizing a product comprising irradiating the product with gammairradiation at a rate from about 0.1 kGy/hr. to about 3.0 kGy/hr. for aperiod of time sufficient to sterilize the product.

[0011] The rate of irradiation can be specifically from about 0.25kGy/hr. to about 2.0 kGy/hr., more specifically from about 0.5 kGy/hr.to about 1.5 kGy/hr. and even more specifically from about 0.5 kGy/hr.to about 1.0 kGy/hr.

[0012] The term “sterilize” as used in the present application generallymeans to inactivate any biological contaminant present in the product.

[0013] The length of time of irradiation or the total dose ofirradiation delivered will depend on the bioburden of the product, thenature of the contaminant and the nature of the product.

[0014] Higher doses of irradiation are required to inactivate viruses ascompared to bacteria. For example, using the dose rates of the presentinvention, one may use an irradiation time of greater than 10 hours toeliminate viral contamination in contrast to an irradiation time of only45 minutes to remove bacterial contamination.

[0015] The process according to the present invention can be carried outat ambient temperature and does not require the heating, freezing,filtration or chemical treatment of the product before the process iscarried out. This offers another significant advantage of the presentprocess as it avoids some of the extra treatment steps of the prior artprocesses.

[0016] Certain products, such as blood, may be diluted prior toirradiation. Diluting the product may serve to reduce degradation of theproduct during irradiation. The choice of diluent depends on the natureof the product to be irradiated. For example, when irradiating bloodcells one would choose a physiologically acceptable diluent such ascitrate phosphate dextrose.

[0017] In cases where living cells (such as blood cells) are to beirradiated, a scavenger may be added to bind free radicals and othermaterials that are toxic to cells. A suitable scavenger is ethanol.

[0018] The efficacy of the method of the present invention is contraryto what others skilled in this area have observed or predicted. (U.S.Pat. No. 4,620,908 and Susan Leitman, ibid). The method provides amethod of irradiating products that is not harmful to the productitself. In particular, the method of the present invention caneffectively sterilize a product as fragile as blood without destroyingthe viability of the cells contained therein. Consequently the method ofthe present invention offers a significant technical and scientificadvancement to the sterilization field. The method also provides aninvaluable service to health care and the general public by providing amethod to produce safe and sterile products for human use. This isespecially critical in light of the spread of viral diseases such asAIDS and hepatitis through the transfusion of contaminated blood andblood products.

DESCRIPTION OF PREFERRED EMBODIMENTS

[0019] The following examples are provided in order to illustrate themethod of the present invention and are not meant to limit the scope ofthe invention.

EXAMPLE 1

[0020] Sterilization of Blood

[0021] A 200 ml-bag of one day old packed red blood cells was used.Ethanol was added to the cells in order to achieve a final ethanolconcentration of 0.01% v/v The red blood cells were diluted by a factorof one in ten using a modified Citrate Phosphate Dextrose (CPD) solutionhaving a pH of about 6.4 to 6.7 and having the following composition ina total volume of 500 ml: Citric Acid Monohydrate  0.2 g Sodium CitrateDihydrate 26.3 g Sodium Monobasic Phosphate  2.2 g Sodium DibasicPhosphate  1.0 g Dextrose  3.2 g

[0022] The cells were irradiated in a commercial size gamma irradiatorwhich contained a cobalt 60 source rack. Irradiation was done offcarrier in an unprotected box. The cells were irradiated for twenty fourhours at a rate of approximately 1 kGy/hr. After the irradiation periodthe red blood cells were examined visually and were found to be viable,having a brilliant red colour. A control sample, consisting of packedred blood cells that were not diluted with the above-described CPDsolution, was not viable after irradiation.

[0023] Four days after the irradiation procedure, the diluted cells weretested for levels of various blood components and the results are shownin Table 1. The control sample consisted of blood from the same bag asthe test sample but it did not undergo irradiation. Table 1 illustratesthat dilution and irradiation of human blood cells did not significantlyalter the white blood cell count. The platelet count and hematocritvalues were slightly lower than the control; however, these values arestill within the range that is seen in normal adult blood. The level ofhemoglobin was higher than in the control indicating that some red bloodcells did lyse during the procedure. This is also evidenced by the lowerred blood cell count. Nevertheless, contrary to what has been previouslypublished, up to 25 kGy of radiation did not destroy the components ofblood by the present procedure. The cells were also counted and found tobe viable after 25 kGy of gamma irradiation delivered at a low dose rateof 1 kGy/hr. TABLE 1 Component Irradiated Blood Control Blood WhiteBlood Cells 4 K/mm³ 4.8 K/mm³ Red Blood Cells 3 Mi/mm³ 7.2 Mi/mm³Hemoglobin 42 g/dl 21 g/dl Hematocrit 46% 64% Platelet 100 k/mm³ 120k/mm³

EXAMPLE 2

[0024] Sterilization of Dextrose

[0025] Dextrose (or glucose) containing solutions are used in thetreatment of carbohydrate and fluid depletion, in the treatment ofhypoglycaemia, as a plasma expander, in renal dialysis and to counteracthepatotoxins (The Merck Index, Eleventh Edition; Merck & Co. Inc.,1989-and Martindale's Extra Pharmacopecia p. 1,265). Dextrose is alsothe preferred source of carbohydrate in parental nutrition regimens (TheMerck Index, Eleventh Edition; Merck & Co. Inc., 1989 and Martindale'sExtra Pharmacopecia p. 1,265). In all of the above applications, thedextrose must be sterilized before use. Sterilization of dextrosecontaining products is generally done by heat sterilization orautoclaving. Unfortunately, these methods have been reported to degradeor caramelize dextrose containing solutions resulting in a colour changein the solution (Martindale's Extra Pharmacopecia p. 1,265). Gammairradiation of glucose has also been reported to decompose glucosecontaining solutions (Kawakishi et al.; Radiation-Induced Degradation ofD-glucose in Anaerobic Condition. Agric. Biol. Chem. June, 1977)Therefore, there is a need for a method that can sterilize dextrosecontaining products that does not degrade the product itself. In view ofthe problems of the prior art, a dextrose solution was treated accordingto the method of the present invention as follows.

[0026] A 5% dextrose solution was irradiated for 24 hours, at a rate ofapproximately 1 kGy/hr. After irradiation the product was tested and itwas found that there was no visible light spectrum change as compared tothe non-irradiated control. Therefore, the present method can be usefulin sterilizing products that contain dextrose.

[0027] In addition to the above experiment, fresh solutions of 5% and50% dextrose were irradiated to 25 kGy over 36 hours at ambienttemperature. The results were similar to those described above. Inaddition, UV/VIS scans were obtained and demonstrated a complete absenceof the peak at 283.4 nm for “furfurals” as per U.S.P. In contrast,dextrose samples sterilized using an autoclave contain the 283.4furfural peak. “Furfurals” are carcinogenic.

EXAMPLE 3

[0028] Sterilization of Human Serum Albumin

[0029] Normal Human Serum Albumin was irradiated as a 25% salt-poorsolution to a total dose of 25 kGy over 36 hours using a Gammacell 220(Co⁶⁰ is the gamma ray source in this instrument). The temperature wasnot controlled during the irradiation but it is estimated that thecontainer holding the albumin solution was approximately 23° C. Theresults of HPLC analysis are given in Table 2. TABLE 2 PARAMETER CONTROL(%) IRRADIATED (%) Polymer 2 3 Dimer 7 8 Monomer 90 86 Low MolecularWeight 1 3 pH 7.05 6.97 NTU (must be > 20) 11.4 11.4

[0030] As the results demonstrate, Normal Human Serum Albumin can safelybe irradiated to 25 kGy (at a rate of approximately 0.7 kGy/hr.) at roomtemperature without adversely affecting the essential properties of theprotein. This has not been demonstrated before. All other attempts atirradiating serum albumin require that it be irradiated in the frozenstage. This adds to the cost and difficulty of doing the irradiation.

EXAMPLE 4

[0031] Normal human blood from a healthy donor was taken in aheparinized tube, washed three times with standard CPD solution, thendiluted 1:20 with CPD containing 0.01% v/v Ethanol. This latter solutionof CPD with 0.01w v/v Ethanol is called SCPD. Two ml aliquots were thenplaced in 10 ml plastic test tubes and irradiated to different doses upto 26 kGy over 36 hours at room temperature. There was no haemolysis andthe cells appeared intact if somewhat large and slightly irregular inshape. The results of three separate experiments are reported in Table3. TABLE 3 PARAMETER RBC¹ HGB² HCT³ MCV⁴ MCH⁵ MCHC⁶ RDW⁷ FLAGS  1* 1.0841 .097 89.5 38.3 427 17.7 Nearly Normal CONTROL .99 33 .089 90.2 33.0366 15.3  2* 95.0 32.3 339 12.0 12 kGy 1 1.22 45 .166 135.8 36.5 26927.3 1 + Anisocytosis 1.38 45 .199 144.7 33.0 228 24.9 3 + Macrocytocis1 1.04 32 .169 163.0 31.3 192 18.8 1 + Anisocytosis 16 kGy 0.54 29 .088162.5 54.5 335 18.8 3 + Macrocytocis 2 0.82 27 .128 156.5 32.8 209 19.82 + Anisocytosis 0.81 26 .124 152.6 32.4 212 20.2 3 + Macrocytocis 10.79 244 .125 158.4 30.8 194 19.4 1 + Anisocytosis 20 kGy 1.26 28 .203161.5 22.1 137 19.0 3 + Macrocytocis 2 0.93 30 .141 151.5 32.3 213 20.12 + Anisocytosis 0.92 30 .143 155.5 32.1 207 20.5 3 + Macrocytocis 26kGy 1 1.15 34 .180 155.9 29.4 189 19.3 1 + Anisocytosis 1.15 34 .176153.0 29.9 195 23.4 3 + Macrocytocis

[0032] The cells were easily put into suspension and reconstituted infresh buffer.

[0033] The following three experiments (Examples 5, 6 and 7) wereconducted in order to determine the efficacy of the method when treatingHIV-contaminated blood. In each Example the cells were similarlytreated. In these experiments, the cells were gently agitated after 12,16 and 24 hours of irradiation. Further, in the third experiment,(Example 7), the cells were placed in T25 flasks to provide greatersurface area and reduce the concentration due to settling in the bottomof the centrifuge tubes. In each case, the cells were irradiated at adose rate of approximately 0.7 kGy/hr.

EXAMPLE 5

[0034] Sterilization of HIV-Containing Blood

[0035] The following experiment was undertaken with the followingspecific objectives:

[0036] 1. To evaluate the toxicity of the process towards red bloodcells (RBCs).

[0037] 2. To evaluate the anti-retroviral activity of the process.

[0038] Procedure

[0039] Initially, 2 ml of anticoagulated blood was obtained from anHIV-seronegative donor. The blood was centrifuged, and the plasma wasremoved. The remaining cell pellet was resuspended in 10 ml of the CPDbuffer and centrifuged. This washing process was repeated a total ofthree times. The final pellet was resuspended in 40 ml of the SCPDbuffer, and distributed into plastic tubes in 2 ml aliquots, with 16separate aliquots being retained for further manipulation. For 8 ofthese tubes, an aliquot of HTLV-IIIB was added. This is a laboratorystrain of the HIV virus and 100 tissue culture infective doses (TCID)were added to each of the tubes to be infected. For the remaining 8tubes, a “mock” infection was performed, by adding a small amount ofnon-infectious laboratory buffer, phosphate buffered saline (PBS). Fourinfected and four non-infected tubes were subjected to the process. Forcomparison, the remaining 8 tubes (four infected and four non-infected)were handled in an identical manner, except that they were not subjectedto the process.

[0040] It should be stated that at the beginning of the study, aseparate aliquot of blood was obtained from the donor. This wasprocessed in the clinical hematology laboratory and a complete hemogramwas performed. These baseline results were compared to repeat testing onthe study aliquots, which included evaluation of four processed and fourunprocessed samples, all of which were not infected with HIV.

[0041] An aliquot of 0.5 ml of each of the infected study samples wasinoculated on mononuclear cells (MCs) which had been obtained three daysearlier. These cells had been suspended in RPMI culture medium, with 10%fetal calf serum and other additives (penicillin, streptomycin,glutamine and HEPES buffer) along with 1 ug/ml PHA-P. At the same timeas this inoculation, the cells were resuspended in fresh medium withrIL-2 (20 U/ml). The cultures were maintained for 7 days. Twice weekly,a portion of the culture medium was harvested for the measurement of HIVp24 antigen levels (commercial ELISA kit, Coulter Electronics, Hialeah,Fla.) for the measurement of viral growth.

[0042] A separate aliquot of the eight infected study samples was usedfor viral titration experiments. Briefly, serial four-fold dilutions ofthe virus-containing fluids (ranging from 1:16 to 1:65,536) wereinoculated in triplicate in 96-well flat-bottom tissue culture plates.PHA-stimulated MCs were added to each well (4 million cells in 2 mlculture medium, with IL-2). An aliquot of the supernatant from eachculture well was harvested twice weekly for the measurement of HIV p24antigen levels. A well was scored as “positive” if the HIV p24 antigenvalue was >30 pg/ml.

[0043] The viral titer was calculated according to the Spearman-Karbermethod (see ACTG virology protocol manual) using the following equation:

M=xk+d[0.5−(1/n)r]

[0044] M: titer (in log 4)

[0045] xk: dose of highest dilution

[0046] d: space between dilutions

[0047] n: number of wells per dilution

[0048] r: sum of total number of wells

[0049] Results

[0050] Red blood cell parameters for the baseline sample as well as forthe unprocessed and processed study samples are shown in Table 4. TABLE4 Sample/Number MCV MCH MCHC Baseline 94.5 32.0 339 Unprocessed-1 91.434.4 376 Unprocessed-2 90.2 37.9 420 Unprocessed-3 92.1 40.0 433Unprocessed-4 91.0 40.2 442 Processed-1 133.4 37.8 284 Processed-2 131.545.0 342 Processed-3 128.5 38.9 303 Processed-4 131.1 39.4 301

[0051] The abbreviations used in Table 4 are explained under Table 3.

[0052] As described above, HIV cultures were established using 0.5 mlaliquots of unprocessed and processed study samples. P24 antigen levels(pg/ml) from the study samples on day 4 and day 7 of culture are shownin Table 5. TABLE 5 Sample/Number p24-DAY 4 p24-DAY 7 Unprocessed-1 1360464 Unprocessed-2 1180 418 Unprocessed-3 1230 516 Unprocessed-4 1080 563Processed-1 579 241 Processed-2 760 303 Processed-3 590 276 Processed-4622 203

[0053] Finally, one unprocessed sample and one processed sample wereselected for the performance of direct viral titration without culture.The results are shown in Table 6. TABLE 6 Sample/Number Titer (log 10ml) Unprocessed-1 1.5 Processed-1 0.0

[0054] The red blood cells were minimally affected by the process,although some reproducible macrocytosis was observed. Although onco-culturing of processed samples, there appeared to be some residuallive virus, this was not confirmed by direct titration experiments.

EXAMPLE 6

[0055] The objective of this experiment was to evaluate the toxicity ofthe process towards red blood cells in a comprehensive manner.

[0056] Methods

[0057] For this experiment, 1 ml of anticoagulated blood was obtainedfrom the same HIV-seronegative donor as in the first experiment. Theblood was centrifuged and the plasma was removed. The remaining cellpellet was resuspended in 10 ml of the CPD buffer and centrifuged. Thiswashing process was repeated a total of three times. The final pelletwas resuspended in 20 ml of the SCPD buffer, and distributed intoplastic tubes in 2 ml aliquots, with all 10 aliquots being retained forfurther manipulation. Eight tubes were subjected to the process, whilethe final two tubes were retained as control, unprocessed tubes. Afterthe processing, all ten tubes were centrifuged, and the resulting pelletwas resuspended in 100 ul buffer. A complete hemogram was performed onthese reconcentrated study samples.

[0058] As in the first experiment, a separate aliquot of blood wasobtained from the donor when the study sample was taken. A completehemogram was performed on this baseline sample. As the study sampleswere re-concentrated to 33-50% of their original state, more directcomparisons with the baseline sample could be undertaken than werepossible in our earlier experiment.

[0059] Results

[0060] Red blood cell parameters for the baseline sample as well as forthe unprocessed and processed study samples are shown in Table 7. Theabbreviations used in Table 7 are defined in Table 3. TABLE 7Sample/Number RBC HGB MCV MCH MCHC Baseline 4.76 152 94.9 31.9 336Unprocessed-1 0.99 33 90.2 33.0 366 Unprocessed-2 1.08 41 89.5 38.3 427Processed-1 1.15 34 153.0 29.9 195 Processed-2 1.15 34 155.9 29.4 189Processed-3 1.26 28 161.5 22.1 137 Processed-4 0.79 24 158.4 30.8 194Processed-5 0.54 29 162.5 54.5 335 Processed-6 1.04 32 163.0 31.3 192Processed-7 1.35 45 144.7 33.0 228 Processed-8 1.22 45 135.8 36.5 269

[0061] There was macrocytosis of the cells which was present in all theprocessed samples. Comparable hemoglobin levels were measured in theunprocessed and processed samples. The absolute values were appropriatefor the residual dilution. The red blood cells are preserved.

EXAMPLE 7

[0062] The objective of this experiment was to verify and expand uponthe results obtained in Example 6.

[0063] Methods

[0064] For this experiment 5 ml of anticoagulated blood was obtainedfrom the same HIV-seronegative donor as in the first two experiments.The blood was centrifuged, and the plasma was removed. The remainingcell pellet was resuspended in 100 ml of the CPD buffer, andcentrifuged. This washing process was repeated a total of three times.The final pellet was resuspended in 100 ml of the SCPD buffer, anddistributed in 25 ml aliquots, in T25 tissue culture flasks, with allfour aliquots been retained for further manipulation. Two flasks weresubject to the process, while the other two were retained as control,unprocessed flasks. After the processing, the contents of each of theflasks was observed and a visual determination of the cells capacity toabsorb oxygen (turning a brighter red on exposure to ambient air) wasmade. Following this, the contents of the flasks were aspirated andcentrifuged, with the residual pellet resuspended in a small volume ofbuffer. A complete hemogram was performed on these re-concentrated studysamples.

[0065] As in Examples 5 and 6, a separate aliquot of blood was obtainedfrom the donor when the study sample was taken. A complete hemogram wasperformed on this baseline sample. As the study samples werere-concentrated to 33-50% of their original state, direct caparisons ofa number of specific parameters would be possible with the baselinesample.

[0066] Results

[0067] On visual inspection, there were no appreciable differencesbetween the processed and unprocessed study samples. Specifically, thereappeared to be a uniform distribution of well suspended cells. Onexposure to ambient air, the contents of all flasks became somewhatbrighter red. No specific quantitative measurements of oxygenation weremade.

[0068] Red blood cell parameters for the baseline sample as well as forthe unprocessed and processed study samples are shown in Table 8. Theabbreviations used in Table 8 are defined under Table 3. TABLE 8Sample/Number RBC HGB MCV MCH MCHC Baseline 4.75 153 95.0 32.3 339Unprocessed-1 0.93 30 151.5 32.3 213 Unprocessed-2 0.92 30 155.5 32.1207 Processed-1 0.82 27 156.5 32.8 209 Processed-2 0.81 26 152.6 32.4212

[0069] This experiment was designed to more closely approximateconditions of red blood cells to be transfused into a patient, and wasconsequently conducted at higher volumes. On a preliminary basis, itdoes not appear that the process impairs the red blood cells' ability tocarry oxygen, although this should be measured more formally.Interestingly, in this experiment, there was no difference in cell sizebetween the processed and unprocessed samples, both being large comparedto baseline. Comparable haemoglobin levels were measured in all thestudy samples.

EXAMPLE 8

[0070] In this experiment, Immunoglobulin G (IgG) was irradiated inlyophilized form.

[0071] Method

[0072] The IgG was irradiated as a powder to a total dose of 25 kGyusing a Gammacell 220. The temperature of the container holding thematerial was approximately 23° C. The dose rate was 0.72 kGy/hr.

[0073] Results

[0074] The results of HPLC analysis of IgG are given in Table 9. As theresults demonstrate, the product appears to be unaffected after beingirradiated to a dose of 25 kGy at room temperature when the irradiationis delivered at a rate of approximately 0.7 kGy/hr. This has not beenpreviously demonstrated. TABLE 9 PARAMETER CONTROL (%) IRRADIATED (%)Polymer (must be >2%) 1 1 Dimer 10 13 Monomer 88 84 Low Molecular Weight1 2

[0075] The results presented by Gergely, et al. using freeze dried IgGshowed that a portion of the protein was insoluble after an irradiationdose of 12 kGy to 25 kGy at standard irradiation dose rates. (Gergely,J., Medgyesi, G. A., Igali, A. Studies of Gamma-Ray-Irradiated HumanImmunoglobulin G. SM-92/12 I.A.E.A.). These results would indicate achange/degradation of the protein. In contrast, using the present methodat a dose rate of approximately 0.7 kGy/hr., none of the protein wasinsoluble. This would indicate that little or no change or degradationof the protein occurred. Further, Gergely et al. found that a liquidformulation of human IgG lost all of its activity after irradiation. Instudies using the present method on intravenous immunoglobulin (IVIG) inliquid form, it was shown that greater than 70% of a specific antibodyin hyperimmune IVIG was retained.

EXAMPLE 9

[0076] In this experiment, alpha 1 proteinase inhibitor and fibrinogenwere irradiated in lyophilized form.

[0077] Method

[0078] The samples were placed in a Gammacell 220 and irradiatedaccording to the present process to a total dose of 25 kGy. Samples werethen returned to the laboratory for analysis. The dose rate was 0.72kGy/hr.

[0079] Results

[0080] The alpha 1 proteinase inhibitor, both treated and control, were40% of a standard normal pooled plasma sample. The Mancini radialimmunodiffusion technique was used as the assay.

[0081] The topical fibrinogen complex vials were reconstituted in 10 mlof water. Protamine sulphate at a concentration 10 mg/ml was added tothe samples. There was instant formation of monomer in all threepreparations.

EXAMPLE 10

[0082] In this experiment, Factors VII, VIII and IX were irradiated inlyophilized form.

[0083] Method

[0084] The samples were placed in a Gammacell 220 and irradiated tovarious total doses at a dose rate of approximately 1 kGy/hr.

[0085] Results

[0086] Factor VII retained 67% activity at 20 kGy and 75% at 10 kGy.Factor VIII retained 77% activity at 20 kGy and 88% at 10 kGy. SimilarlyFactor IX showed an activity level of 70% at 20 kGy and 80% at 10 kGy.

[0087] Analysis

[0088] Excellent results were found for the three Factors. To ourknowledge, no one has been able to achieve these results by irradiatingthe Factors at ambient temperature to such a high dose of radiation withsuch little loss of activity. This is in direct contrast with theresults of Kitchen et. al. (Kitchen, A. D. Mann, G. F., Harrison, J. F.,Zuckerman, A. J. Effect of Gamma Irradiation on the HumanImmunodeficiency Virus and Human Coagulation Proteins. Vox Sang 1989,56:223-229) who found that “the irradiation of lyophilized concentratesis not a viable procedure”. Similarly, Hiemstra et. al., (Hiemstra, H.,Tersmette, M., Vos., A. H. V., Over, J., van Berkel, M. P. and de Bree,H. Inactivation of human immuondeficiency virus by gamma radiation andits effect on plasma and coagulation factors. Transfusion, 1991,31:32-39) also concluded that “Gamma radiation must be disregarded as amethod for the sterilization of plasma and plasma-derived products,because of the low reduction of virus infectivity at radiation dosesthat still give acceptable recovery of biologic activity of plasmacomponents.”

EXAMPLE 11

[0089] In this experiment, red blood cells were irradiated at a doserate of 0.5 kGy/hr. for periods of time ranging from 7½ to 90 minutes inorder to remove bacterial contaminants.

[0090] Method

[0091] Red blood cells were collected from a healthy donor in EDTA,washed 3 times with CPD solution and resuspended in CPD to provide a1:20 dilution based on the original blood volume. The cell suspensionwas then subdivided into 14 tubes. To seven of the tubes approximately1.0×10⁴ Staphylococcus epidermidis were added. The cells were placed onice for transport to the irradiation facility. All of the samples wereplaced in the chamber at ambient temperature and irradiated at 0.5kGy/hr. for periods of time to give total doses of 0.0625, 0.125, 0.250,0.375, 0.500 and 0.750 kGy respectively. The samples were removed andagitated at each time point and placed on ice for transport either tothe microbiology lab or to the hematology lab for analysis.

[0092] Results

[0093] The results of the microbiology assays are given in Table 10.TABLE 10 RADIATION DOSE (kGy) TIME (MIN) NUMBER SURVIVING 0 92200 0.06257.5 84500 0.125 15 35000 0.250 30 10067 0.375 45 1800 0.500 60 250 0.75090 0

[0094] Thus a dose of 0.75 kGy provides a 4.5 log₁₀ reduction inbacterial survivors. This represents a significant safety factor forblood. Further, the D₁₀ value is approximately 0.125 kGy whichcorresponds well with the values reported in the literature for similarspecies of staphylococcus (B. A. Bridges, The effect of N-Ethylmaleimideon the radiation sensitivity of bacteria. J. Gen. Microbiol.1961, (26)467-472 and G. P. Jacobs and N. Sadeh, Radiosensitization ofStaphylococcus aureus by p-hydroxybenzoic acid. Int. J. Radiat. Biol;1982,41,351-356).

[0095] In order to demonstrate that the red blood cells remained viableafter the irradiation process, the following parameters were determinedfor the cells; WBC, Neutrophils, Lymphocytes, Monocytes, Eosinophils andBasophils. These determinations merely enumerated the number of cellspresent. All nucleated cells would, of course, be inactivated by theradiation dose delivered. The other red blood cell parameters monitoredare listed in Table 11. The Methaemoglobin value was unchanged from thatof the controls even after a radiation dose of 0.75 kGy. This experimentdemonstrates that red blood cells can be safely irradiated by thepresent method to a dose of 0.75 kGy at room temperature with no loss ofcell function.

[0096] Red Blood Cell Values as a Function of Radiation Dose ReceivedTABLE 11 Whole Total Dose (In kGy) Parameter Blood 0 0.0625 0.125 0.2500.500 RBC 5.06 1.49 1.27 1.77 1.73 1.43 HGB 153 43 41 56 56 46 HCT .483.142 .120 .167 .163 .131 MCV 95.5 95.6 94.3 94.2 93.7 92.1 MCH 31.2 31.132.2 31.7 32.2 32.5 MCHC 327 325 341 336 344 353 RDW 13.3 12.1 12.7 12.912.9 13.2 METHgB 0.9 0.3 0.3 0.3 0.0 0.9

EXAMPLE 12

[0097] This experiment was conducted using the method in Example 11 toconfirm the findings of Example 11 and to expand upon some of theparameters measured. The results of this experiment are given in Table12.

[0098] Results

[0099] Red Blood Cell Values as a Function of Radiation Dose ReceivedTABLE 12 Total Dose (In kGy) PARAMETER 0 0.0625 0.125 0.250 0.375 0.5550.750 HGB 1.8 1.7 1.8 1.7 2.0 2.0 2.0 % O 96.6 96.5 96.2 96.3 96.4 96.596.0 % CO 1.0 1.2 1.6 1.3 1.7 1.5 1.5 % MET 0.5 0.5 −0.5 0.4 −0.2 0.40.8 % REDUCED 1.9 1.9 2.7 2.2 2.2 1.7 1.7 p60 (mm Hg) 34 nd nd nd nd nd24 Hill 2.1 nd nd nd nd nd 1.8 Coefficient

[0100] These results confirm the previous results and indicate thatindeed, red blood cells can be irradiated to a dose sufficient toprovide 4.5 log₁₀ reduction in bacteria count.

[0101] It is contemplated that future experiments will provide similarresults for platelets. Thus with little or no additional manipulation,and without the addition of extraneous materials, red blood cells can betreated by the present process to provide a bacteriologically safeproduct, thus further reducing the risk of untoward reactions inrecipients.

[0102] As evidenced by all of the above experiments, the presentinvention demonstrates that irradiating a product at a low dose ratefrom about 0.1 kGy/hr. to about 3.0 kGy/hr. is effective in sterilizingthe product without adversely affecting the product itself.

[0103] While the Examples relate to specific embodiments of the methodof the present invention, one skilled in the art will realize that thetotal time of irradiation will depend on the type of contaminant, thebioburden of the product and the nature of the product. For example,bacterial contaminants can be eliminated with very little irradiationtime while viral inactivation requires a longer irradiation time.Further, extremely sensitive products, such as blood, are preferablydiluted in a physiologically acceptable diluent prior to irradiation.

[0104] It is to be appreciated that the method of the present inventioncan be used to treat an extremely wide variety of products that requiresterilization. The fact that the present method has proven effective inblood which is a fragile biological material makes it reasonable topredict that the method can be used on many similarly sensitiveproducts. Examples of other products that may be treated includepharmaceuticals, proteins, nucleic acids, blood components, body fluids(such as cerebral spinal fluid, saliva), liposomes, glucose containingproducts, cell culture media, fetal bovine serum, bone marrow, organs,foods and cosmetics such as shampoos, lotions and creams. The productsmay be irradiated in various forms, including, solid, liquid andlyophilized forms.

What i claim as my invention is:
 1. A method for sterilizing a productcomprising irradiating the product at ambient temperature with gammairradiation at a rate from about 0.1 kGy/hr. to about 3.0 kGy/hr. for aperiod of time sufficient to sterilize the product.
 2. A methodaccording to claim 1, wherein said irradiation is provided at a rate offrom about 0.25 kGy/hr. to about 2.0 kGy/hr.
 3. A method according toclaim 1, wherein said irradiation is provided at a rate of from about.0.5 kGy/hr. to about 1.5 kGy/hr.
 4. A method according to claim 1,wherein said irradiation is provided at a rate of from about 0.5 kGy/hr.to about 1.0 kGy/hr.
 5. A method according to claim 1, wherein saidproduct is an organic product.
 6. A method according to claim 1, whereinsaid product is a biological product.
 7. A method according to claim 1,wherein said product is blood or a component thereof.
 8. A methodaccording to claim 7, wherein said blood or blood component is firsttreated with ethanol.
 9. A method according to claim 8, wherein saidethanol is in a final concentration of approximately 0.01% to 0.05% v/vand said blood or blood product is diluted before irradiation in aphysiologically acceptable diluent to achieve a final dilution of atleast 1:10.
 10. A method according to claim 9, wherein saidphysiologically acceptable diluent is a modified citrate phosphatedextrose solution having a pH in the range of about 6.4 to about 6.7.11. A method according to claim 10, wherein said citrate phosphatedextrose solution contains about 0.01% v/v ethanol.
 12. A methodaccording to claim 7, wherein said product is diluted with a citratephosphate dextrose solution.
 13. A method according to claim 1, whereinsaid product contains dextrose.
 14. A method according to claim 1,wherein said product is a protein.
 15. A method according to claim 14,wherein said product is an antibody.
 16. A method according to claim 1,wherein said product is in lyophilized form.
 17. A method according toclaim 1, wherein said product is selected from the group consisting ofIgG, albumin, alpha 1 proteinase inhibitor, fibrinogen, Factor VII,Factor VIII and Factor IX.
 18. A method according to claim 16, whereinsaid product is selected from the group consisting of IgG, albumin,alpha 1 proteinase inhibitor, fibrinogen, Factor VII, Factor VIII andFactor IX.